Compelling new data suggests that e-cigarettes are toxic to the lungs. Indeed, there have now been over 700 hospitalization cases due to acute lung injury in vapers. The cause(s) of this injury are poorly understood and whilst many hospitalized vapers said that they have used THC diluted in Vitamin E oil, many did not. We previously collected serum, saliva and sputum from healthy never-smokers, tobacco smokers or vapers. We also performed research bronchoscopies and collected bronchoalveolar lavage fluid (BALF). From these samples, our group found unique changes to the bronchial and secreted proteomes. We also found that vaping increased levels of lung proteases that are known to cause emphysema and bronchiectasis. Our preliminary data indicate that vapers? macrophages are phenotypically different to non-smokers? and smokers? macrophages, have a greater M1/pro-inflammatory phenotype and significant downregulation of gene expression. Moreover, our new data indicate that vapers? macrophages have increased numbers of intracellular vacuoles, which may be caused by lipid accumulation. Since we see extensive and significant changes in the vaper?s lung samples, even though they are ?healthy?, we propose that they are an ideal biobanked data set that can be used to assess the extent to which altered lung lipid content and lipid-laden/foamy macrophages exist. Importantly, since we obtained an NIH certificate of confidentiality, we also know about any potential drug use. We have recently developed assays to detect nicotine in vapers? BALF and sputum and we also propose to develop our analytic techniques in order to detect THC and its metabolites in these samples, and we will correlate any changes in lung macrophages/lipids with lung nicotine and THC levels. To achieve these goals, we propose the following specific aims: Aim 1. To determine the concentrations of surfactant proteins in biobanked samples of BAL, sputum and saliva from non-smokers, smokers and vapers. Aim 2. To perform lipidomics and stain alveolar macrophages with Oil Red O to look for altered lipid content in vaper?s macrophages and airway secretions. Aim 3. To perform metabolomics on biobanked samples of BAL, sputum and saliva from non-smokers, smokers and vapers. These studies will use biobanked lung samples from a CTP-funded study (P50 HL120100). We believe that this proposal is responsive to the following FDA areas of interest: (i)Examine health outcomes from various tobacco product types, not to include cigarettes; (ii) Examine the long- and short-term health effects of transitioning from combustible to non-combustible tobacco, and from non-combustible to combustible tobacco, using biomarker and/or health outcomes; (iii) Compare biomarker expression profiles with self-reported use of tobacco products, and analyze measures associated with biomarkers of exposure that may inform risk associated with tobacco exposure for mortality/morbidity by age, sex, and race/ethnicity.